Systems and methods for Brillouin spectroscopy and tissue imaging - Patents.com

Abstract

Systems and methods are provided for measuring the mechanical properties of ocular tissue, such as lens or corneal tissue, for both diagnostic and treatment monitoring purposes. A laser-locked feedback system is provided to achieve frequency accuracy and sensitivity that facilitates surgery and diagnosis with superior sensitivity and precision. Differential comparison between ocular tissue regions in either the same eye or the other eye of a patient can further facilitate early diagnosis and monitoring. [Selected Figure] Figure 1

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 547,171, filed August 18, 2017, and entitled "APPARATUS FOR BRILLOUIN SPECTROSCOPY AND IMAGING OF TISSUES."

[0002] <Statement Regarding Federally Sponsored Research> none. [Background technology]

[0003] FIELD OF THE DISCLOSURE The present disclosure relates to tissue imaging, and more particularly to improved systems and methods for Brillouin spectroscopy and / or Brillouin microscopy.

[0004] The elasticity of a normal, healthy cornea is typically uniform throughout the volume of the corneal tissue. "Corneal ectasia" refers to the protrusion of the cornea, which occurs when it is not mechanically strong enough to withstand intraocular pressure. Although ectasia is a rare condition, it is also one of the serious adverse consequences that occur after LASIK (laser in situ keratomileusis) surgery, causing a thinning or weakening of the cornea. In this context, pellucid marginal degeneration (PMD) is typically characterized by thinning of the inferior and peripheral regions of the cornea in one or both eyes. Keratoconus is also a disease characterized by corneal thinning. These and other conditions can be associated with localized weakening or thinning of ocular tissue and localized differences in biomechanical properties, such as elasticity. The biomechanical properties of ocular tissue can also be suitable targets for the diagnosis and monitoring of the onset and progression of cataracts and presbyopia, as well as for corneal pathology and treatment.

[0005] As is evident from the above, the biomechanical properties of ocular tissues are relevant to several diseases and refractive procedures. Accordingly, various techniques have been developed to assess the biomechanical properties of ocular tissues.

[0006] However, the sensitivity and specificity of biomechanical measurements are often compromised by confounding factors: for example, the accuracy and sensitivity of air-puff corneal stiffness measurements using Scheimpflug topography or optical coherence tomography can be significantly reduced by the effects of intraocular pressure (IOP), and accurate measurement of IOP can be compromised by coupling between IOP measurements and corneal stiffness.

[0007] In Brillouin imaging, the Brillouin scattering properties of tissues are sensitive to the tissue hydration level. This can lead to ambiguity in interpreting tissue stiffness from the measured Brillouin shift, as corneal hydration has diurnal and individual variations that can lead to large variability and confound clinical interpretation. Another confounding factor in Brillouin spectroscopy is tissue temperature. The Brillouin frequency shift of tissues can vary with temperature. Thus, Brillouin measurements taken at a certain body temperature may give different values ​​compared to measurements taken at another body temperature (e.g., due to fever), which can lead to misinterpretation. For example, the temperature dependence of the Brillouin frequency shift of tissues can be approximately 7.45 MHz / °C for an optical wavelength of 780 nm.

[0008] Of further concern, good Brillouin frequency sensitivity and accuracy within ±10 MHz is required to distinguish subtle changes in tissue biomechanical properties or detect abnormalities at early stages of disease. The accuracy of traditional Brillouin spectroscopy or microscopy systems can be significantly compromised by frequency drift of free-running laser sources or temperature changes in the environment, which can cause thermodynamic shifts of components. For example, the frequency drift of a typical external cavity semiconductor laser is about 100 MHz over a 10 minute duration, even in a temperature-controlled room. Typically, the laser output contains a high level of background spontaneous emission (-50 to -55 dB) relative to the stimulated emission laser line. This background noise is particularly problematic in Brillouin microscopy, because backreflected light from the optics or tissue surface enters the Brillouin spectrometer, and this backreflected light is broadband in nature and difficult to separate from the weak Brillouin signal.

[0009] Thus, while currently existing biomechanical measurement techniques and systems can provide useful and valuable information for evaluating patient tissue, further improvements are desirable. Summary of the Invention

[0010] The present disclosure generally provides novel and useful systems and methods for measuring mechanical properties of tissue, such as lens-corneal tissue, for diagnostic and treatment monitoring purposes. In one aspect, the present disclosure uses a laser-locked feedback system that can achieve better frequency accuracy and sensitivity than conventional Brillouin spectroscopy or microscopy systems. In another aspect, a novel Brillouin imaging method is provided that uses differential comparison between multiple ocular tissue regions in either the same eye or the other eye of a patient to produce improved results. In some cases, a unique human interface configuration can be used to measure biomechanical properties at any desired location (e.g., in the xy plane) and / or at any desired depth (e.g., along the z axis) within the tissue volume.

[0011] In one aspect, the present disclosure provides a Brillouin spectroscopy system for evaluating tissue at an ocular tissue region of a patient, the system comprising a laser source system including a tunable laser source configured to generate a first electromagnetic radiation having an electromagnetic spectrum and a vapor cell reference configured to capture a portion of a polarized form of the first electromagnetic radiation and generate an error signal including information regarding a deviation of the electromagnetic spectrum from a target electromagnetic spectrum. The vapor cell reference includes a vapor cell configured to receive the portion of the first electromagnetic radiation and selectively transmit the portion of the first electromagnetic radiation based on the electromagnetic spectrum, and a detector configured to receive the transmitted portion of the first electromagnetic radiation and generate an error signal. The Brillouin spectroscopy system also includes a human interface configured to transmit the first electromagnetic radiation to the ocular tissue region of the patient, the first electromagnetic radiation generating at least one acoustic wave at the ocular tissue region, and at least one second electromagnetic radiation being generated based on the at least one acoustic wave. The Brillouin spectroscopy system further includes a spectrometer system configured to receive a portion of the second electromagnetic radiation to provide information related to a biomechanical property of the ocular tissue region.

[0012] In another aspect of the present disclosure, a laser source system for generating a laser with a stabilized peak frequency and filtered spontaneous emission noise is provided. The laser source system includes a tunable laser source configured to generate a first electromagnetic radiation having an electromagnetic spectrum and a vapor cell-based reference unit configured to obtain a polarization form of a portion of the first electromagnetic radiation and generate an error signal including information regarding a deviation of the electromagnetic spectrum from a target electromagnetic spectrum. The vapor cell-based reference unit includes a first polarizer configured to receive the portion of the first electromagnetic radiation and change a polarity of the portion, a vapor cell configured to receive the portion of the polarized form of the first electromagnetic radiation from the first polarizer, a second polarizer configured to receive the portion of the polarized form of the first electromagnetic radiation from the vapor cell and change a polarity of the portion of the first electromagnetic radiation, and a detector configured to receive the portion of the polarized form of the first electromagnetic radiation from the second polarizer and generate an error signal.

[0013] Another aspect of the present disclosure provides a method for evaluating ocular tissue of a patient, the method including obtaining a first biomechanical value of a first ocular tissue region of the patient using Brillouin spectroscopy, obtaining a second biomechanical value of a second ocular tissue region of the patient, and comparing the first biomechanical value to the second biomechanical value to identify a medical condition of the ocular tissue of the patient.

[0014] Another aspect of the present disclosure provides a Brillouin spectroscopy system for evaluating tissue in an ocular tissue region of a patient. The system includes a laser source system including a tunable laser source configured to generate a first electromagnetic radiation having an electromagnetic spectrum and a vapor cell reference configured to capture a portion of the first electromagnetic radiation and generate an error signal including information regarding a deviation of the electromagnetic spectrum from a target electromagnetic spectrum, the error signal being generated by monitoring absorption of the first electromagnetic radiation by atoms in the vapor cell reference. The Brillouin spectroscopy system also includes a human interface configured to transmit the first electromagnetic radiation to the ocular tissue region of the patient, the first electromagnetic radiation interacting with at least one endogenous acoustic wave by the ocular tissue region and at least one second electromagnetic radiation being generated based on the at least one acoustic wave. The Brillouin spectroscopy system further includes a spectrometer system configured to receive a portion of the second electromagnetic radiation to provide information related to a biomechanical property of the ocular tissue region.

[0015] These and other aspects and advantages of the present invention will become apparent from the following description, in which reference is made to the accompanying drawings, which form a part of the specification, and in which there is shown by way of illustration preferred embodiments of the invention, which, however, do not necessarily represent the full scope of the invention, and in which the claims should be consulted in interpreting the scope of the invention. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a block diagram of a Brillouin imaging system according to the present disclosure. [Diagram 2] FIG. 1 is a block diagram of a laser light source system according to the present disclosure. [Figure 3A] FIG. 1 is a schematic diagram showing a two-fiber scheme of the human interface used in the Brillouin imaging system. [Figure 3B]FIG. 1 is a schematic diagram showing a one-fiber overview of the human interface used in the Brillouin imaging system. [Figure 4] FIG. 1 is a block diagram showing the optical path of one fiber schematic of the human interface used in the Brillouin imaging system. [Figure 5A] FIG. 1 is a schematic diagram of an optical probe constituting the fixed focal length pen endoscope disclosed herein; [Figure 5B] 1 is a schematic diagram of an optical probe constituting an adjustable-axis endoscope according to the present disclosure; [Figure 5C] 1 is a schematic diagram of an optical probe constituting a rotational catheter according to the present disclosure. [Figure 6] FIG. 1 is a schematic diagram of a spectrometer equipped with a Rb vapor absorption filter and a single stage VIPA etalon of the present disclosure. [Figure 7] This graph shows the attenuation spectrum (black) of a 10 cm long Rb vapor cell at a vapor pressure of 9 μPa (65° C.), the spectrum of the laser signal to be rejected (702, amplitudes not evenly spaced), and the approximated signals from the corneal stroma (704) and aqueous humor (706, amplitudes not evenly spaced). [Figure 8] FIG. 1 is a simplified block diagram of an example computer system that can be used with the Brillouin imaging system of the present disclosure. [Figure 9] 1 is a flowchart illustrating some example, non-limiting steps of a method of evaluating tissue of a patient's eye disclosed herein. [Figure 10] 1 is a schematic diagram showing several components near the cornea of ​​a human eye and pressure and diffusion arrows. [Figure 11] 1 is a graph showing the Brillouin frequency shift at 780 nm in pure water measured by experiments in the temperature range of 15 to 40° C. [Figure 12A] 1 is a set of interrelated graphs showing experimental results of measured diurnal variations in Brillouin frequency shift and CCT measurements. [Figure 12B] 1 is a graph showing experimental results of measured central corneal Brillouin frequency shift OD-OS difference in 37 patients. [Figure 13] 1 is a set of maps presenting experimental results of the Brillouin frequency shift measured for both eyes of subjects classified as having early mild keratoconus (left set) and normal healthy patients (right set). [Figure 14] 1 is a set of maps presenting experimental results of measured local heterogeneity in patients with early stage keratoconus. [Figure 15A] 1 is a graph showing regional differences in Brillouin frequency shift measurements of the cornea with keratoconus. [Figure 15B] 1 is a graph showing local differences in Brillouin measurements in corneas with stages I to IV keratoconus. [Figure 16] 1 is a graph illustrating a tissue evaluation system of the present disclosure that can be configured to obtain a first biomechanical value of tissue in a patient's eye. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Aspects of the present disclosure encompass ophthalmic systems and methods for data acquisition, processing, and display of biomechanical parameters that incorporate Brillouin spectroscopy or other assessment techniques. Examples of tissue evaluation or assessment techniques that can be used with the systems and methods disclosed herein include, but are not limited to, optical coherence tomography (OCT) modalities, Brillouin imaging modalities, Raman imaging modalities, laser speckle imaging modalities, multiphoton imaging modalities, photoacoustic imaging modalities, confocal microscopy imaging modalities, fluorescence imaging modalities, Pentacam® imaging modalities, ultrasound imaging modalities, and approaches that combine or include one or more of the above imaging modalities. In this regard, examples of tissue evaluation or assessment techniques that can be used with the systems and methods disclosed herein include U.S. Patent Nos. 7,898,656, 8,115,919, 9,777,053, U.S. Patent Application Publication Nos. 2012 / 0302862, and 2016 / 0151202, the contents of which are incorporated herein by reference.

[0018] FIG. 1 illustrates a Brillouin imaging system 100 for assessing biological tissue with improved measurement accuracy and reliability. The system or device 100 may utilize Brillouin spectroscopy and / or Brillouin microscopy or equivalent Brillouin imaging techniques. The system 100 benefits from improved signal filtering to allow probing of highly scattering tissues such as the sclera, skin and blood vessels. The system 100 may include a laser source system 102, a human interface 104, and a spectrometer system 106. In general, without being bound by theory, the laser source system 102 generates electromagnetic radiation that can be delivered to the biological tissue using the human interface 104. The electromagnetic radiation can generate mechanical stress modulation in the tissue via thermal or electrical contraction phenomena. If the stress modulation is phase-matched to one of the characteristic acoustic phonon modes in the tissue, the acoustic phonon mode can be efficiently generated through a coherence process. The excited acoustic phonons can generate refractive index modulation in the medium, resulting in inelastic scattering of photons. The energy and momentum of the photons can be changed by the inelastic scattering process. The spectrometer system 106 can measure the scattered photons. The magnitude of the frequency shift of the scattered photons can be substantially or approximately equal to the magnitude of the frequency shift of the acoustic phonons, and thus biomechanical information of the tissue can be derived. The system 100 can optionally include a computer system 108 that can support a number of functions, including but not limited to, processing information from the spectrometer 108 or providing feedback signals to the laser source system 102 or the human interface 104.

[0019] <Laser light source system>

[0020] The laser source system 102 may comprise a laser capable of emitting a narrowband spectrum locked to a specific absorption line or target wavelength. The laser may have a stabilized peak frequency and filtered spontaneous emission noise. The absorption line may be an atomic species absorption line, such as the rubidium absorption line at 780 nm. The laser may also comprise a vapor cell based reference and / or a multi-stage spectral clean-up filter, which may be locked to the output frequency of the laser.

[0021] 2 is a schematic diagram of a laser source system 202 optimized for Brillouin imaging. The laser source system 202 includes a laser source 222, a vapor cell-based reference 224, and a spectral clean-up filter 226. As shown in the figure, the following abbreviations are used: thermoelectric controller (TEC), Wollaston prism (WP), balanced detector (D1), photodiode (D2), magnet (Mag), and piezoelectric transducer (PZT).

[0022] The laser light source system may include a radiation emission (e.g., light) source 250, which may be a single frequency laser, a filtered mercury lamp, or other type of light source known in the art. The radiation emission source may have a wavelength between about 530 nm and 1350 nm, for example, although other wavelengths known to be safe for use by the eye may be used. The linewidth of the radiation may typically be less than about 1 GHz, or more preferably less than about 100 MHz, although other sources with broader linewidths or multiple spectral lines may be used with appropriate geometry. The radiation source 250 may use an optical arrangement to output multiple frequency lines to enhance the Brillouin scattering signal. The scattered radiation (e.g., light) from the sample may include multiple frequency components resulting from both simple elastic scattering and Brillouin scattering.

[0023] The illustrated light source 250 is a single frequency distributed feedback (DFB) laser. However, the light source 250 can be of another type, such as a grating type or an external cavity diode laser (ECDL). An integrated thermoelectric (ET) controller can be used to control the laser temperature. Since DFB lasers are more sensitive to temperature than free space ECDLs, it may be desirable to control the laser temperature to within about 0.01°C using an integrated thermoelectric controller (TEC) as described above, which has a typical accuracy of about 0.001°C. If the laser is operating at a wavelength of about 780 nm, which coincides with the absorption line of rubidium (Rb) atoms, the output frequency can be locked to the transition peak of rubidium as shown in FIG. 2. For other laser wavelengths, other atomic species can be used.

[0024] The vapor cell reference 224 may include a vapor cell filter 252 configured to receive a portion of the first electromagnetic radiation and selectively transmit the portion of the first electromagnetic radiation based on the electromagnetic spectrum. The vapor cell reference 224 may also include a detector configured to receive the transmitted portion of the first electromagnetic radiation and generate an error signal that includes information about the deviation of the electromagnetic spectrum from a target electromagnetic spectrum. Alternatively, the vapor cell reference 224 may include alternative components that may generate an error signal by monitoring the absorption of the source laser radiation by atoms in a small unheated reference vapor cell. For example, instead of using the polarization dependence of atomic absorption in a magnetic field, a small modulation may be applied to the laser frequency. Although the fraction captured is illustrated as 4% of the radiation generated by the source 250, the fraction captured may be increased or decreased. The laser source system 202 may have a laser locking configuration based on an atomic vapor line that is substantially temperature independent. The vapor cell of the embodiment shown in FIG. 2 may be placed in a weak magnetic field. 85 D of Rb vapor atoms 2 A laser locking technique is used that utilizes the Zeeman effect in a line.

[0025] In one aspect, the vapor cell-based reference unit 224 can include a first polarizer configured to receive the portion of the first electromagnetic radiation and change the polarity of the portion, a vapor cell configured to receive the portion of the polarized form of the first electromagnetic radiation from the first polarizer, a second polarizer configured to receive the portion of the polarized form of the first electromagnetic radiation from the vapor cell and change the polarity of the portion of the first electromagnetic radiation, and a detector configured to receive the portion of the polarized form of the first electromagnetic radiation from the second polarizer and generate an error signal.

[0026] In FIG. 2, a polarized portion of the output from laser 250 enters vapor cell 252. Due to the presence of a weak magnetic field, the absorption curves of the two circularly polarized components are shifted to higher and lower frequencies, respectively. After passing through vapor cell 252, the beam propagates through a quarter-wave plate, after which the beam can propagate through a Wollaston polarizing prism. The diverging curve caused by the difference between the two signals can provide an error signal for frequency locking. This error signal can be sent to computer system 208, which can provide a signal to the thermoelectric controller of the laser source.

[0027] Throughout the experiment, frequency stability of better than 2 MHz of the experimental vapor cell transition line has been observed, and the laser frequency can be actively stabilized to within ±10 MHz using analog servo control over a wide range of ambient temperatures, typically ranging from 15 to 25 °C. Alternatively, frequency locking to a solid-state etalon can be used, but silica etalons are sensitive to temperature (approximately 3.8 GHz / °C) as a frequency reference.

[0028] The spectral clean-up filter 226 can be locked to the output frequency of the laser and can be used to reduce spectral noise. The spectral clean-up filter 226 can have multiple tandem free-space Fabry-Perot (FP) cavities, each of which is comprised of a pair of concave mirrors. These mirrors can have a reflectivity of about 97%. The spectral clean-up filter can be configured to provide an amplified spontaneous emission (ASE) suppression of up to about 30 dB (per cavity) with a low insertion loss of between 1.5-3 dB. The spectral clean-up filter can generate an ASE error signal. This error signal can be sent to the computer system 208, which can provide a signal to the thermoelectric controller of the laser source. Each free-space cavity can be locked to an operating wavelength, or this can be achieved using a piezoelectric transducer (PZT). Frequency locking techniques can be used, such as dither lock or similar configurations. The free spectral range of each tandem FP cavity can be mismatched to achieve background rejection over a wider spectral range around the laser peak. With mirror reflectivity near 90%, about 20 dB rejection can be achieved from each cavity with low loss of less than 0.5 dB. Thus, the attenuation of the tandem cavity can be about 40 dB with loss of less than 1 dB. Using frequency-locked lasers in conjunction with clean-up filters, a high purity output can be produced with laser-to-noise levels of, for example, more than 80 dB.

[0029] The accuracy provided by the vapor cell reference 224 can be improved by using counter-propagating beams to obtain narrower features for locking, or by performing a differential measurement with the two beams, or the like. In one embodiment of the laser source system 202, laser modulation can be used to generate both the laser lock error signal and the cavity (ASE filter) lock error signal.

[0030] <Human Interface>

[0031] The human interface 104 serves as an interface between the output of the laser source system and the subject to direct the generated electromagnetic energy to the target tissue as intended, and also serves as an interface between the scattered electromagnetic radiation and the spectrometer system to ensure that a portion of the scattered electromagnetic energy is measured.

[0032] Typically, the output of the laser source system is sent to a human interface and then to the subject. The scattered light from the subject is collected at the human interface and sent to a spectrometer for analysis. The scattered light contains not only the Brillouin scattering signal but also background noise with the same frequency spectrum as the probe laser. This noise is suspected to come from two main sources: reflections from various optical components of the system and elastic scattering from the tissue. For corneal imaging, Fresnel-type specular scattering at the corneal surface is often several orders of magnitude stronger than Brillouin scattering (corneal reflectance is up to 2% at normal incidence), and this specular reflection must be suppressed. Typically, lenses used in the human interface are preferably anti-reflective coated and are positioned so that the light beam strikes the lens surface at an angle and is oblique to the optical path to minimize back reflections. In addition, the probe beam axis can be oblique to the normal axis of the corneal surface by about 15°.

[0033] In this disclosure, a rejection efficiency of about 65 dB is sufficient for corneal imaging. However, this boundary rejection efficiency limits the selection of optical components and the design of the human interface. As a result, the example human interface system 304 has a bulk optical configuration and requires that the beam enters the objective lens off-axis. This is evident from FIG. 3A. In the example system 304, the following abbreviations are used: polarization-maintaining fiber (PMF), single-mode fiber (SMF), mirror (M), motorized shutter (S), and reference material (Ref). The above-mentioned off-axis requirement precludes simple beam scanning in the lateral directions (X and Y).

[0034] To avoid excessive beam specular reflection from the tissue surface, the incidence angle can be larger than about 5°. Some tissues with layered microstructures, such as corneal tissue with its collagen-rich layers, have anisotropic properties. Therefore, the Brillouin shift value of such tissues depends on the inclination angle of the beam with respect to the tissue surface. This angular dependence can be taken into account in the spectral analysis, providing a novel source of information about the orientation and structure of collagen fibers in suspected pathologies. Angular-dependent metrics can be relatively insensitive to factors such as temperature and tissue hydration.

[0035] Due to the high attenuation of the Rb filter and VIPA etalon, and the improved signal-to-background ratio of the laser source system, a human interface can be constructed with a 2×2 fiber optic coupler or alternatively a fiber optic circulator. The circulator replaces the bulk assembly of the PBS, the wave plate, and the fiber coupling optics. As shown in the example system 314 of FIG. 3B, the human interface can use a two-axis galvanometer mirror scanner and on-axis beam alignment. This single fiber configuration includes a fiber optic circulator and a beam scanner. To avoid specular reflections, the beam incidence angle on the tissue is oblique to the surface normal. System 314 adds a controllable motorized flip mirror (FM).

[0036] Conventional systems include a reference material with a known Brillouin frequency shift in the human interface and a mechanical shutter to calibrate the measured Brillouin frequency shift. The human interface may also include the reference material in a temperature-controlled mount. Alternatively, a temperature sensor may be attached to the reference material, and the temperature-dependent Brillouin shift of the reference material is used for calibration. In another approach, an electro-optic frequency modulator (EOM) can be used to generate precision frequency sidebands in a laser for spectrometer calibration. One advantage of this technique is that a complete calibration curve can be generated by scanning the EOM frequency, thus correcting for nonlinearities in the VIPA emission. The Rb cell system instead uses two abundant isotopes of Rb (e.g. 85 Rb and 87 Several different transitions of Rb around 780 nm (e.g., several D 2 transitions) can be used as calibration points for the spectrometer.

[0037] An infrared light source and a monitoring camera can be used for tracking the beam and the eye. To improve profiling of the corneal surface, structured light surface reconstruction can be added using the same monitoring camera with the addition of an LED pattern projector. A fluorescent dye, such as fluorescein, can be used as an imaging contrast agent. Custom beam registration software can be used with the computer system 108. The computer system can also be used to control the XY scanning portion based on real-time images of the cornea using an algorithm. Z scanning can be achieved by motorized linear movement of the objective lens. The user interface system can provide a uniform grid of scanning points. Additionally, an expanded field of view can be provided to cover the cornea including the limbus. For example, the expanded field of view can be approximately 8 mm x 8 mm.

[0038] FIG. 4 is a schematic diagram of one embodiment of a human interface 414 depicting the optical path of the system corresponding to that shown in FIG. 3B. In the figure, a laser source system 402 can provide a first electromagnetic radiation 410, which can be provided to an eye 420. One example form of the electromagnetic radiation 410 can be light in the visible or near infrared range. The electromagnetic radiation 410 can be sent to the eye 420 to probe different portions of the eye tissue. These different portions include, but are not limited to, the cornea 422 and the crystalline lens 424. For example, an imaging lens 430 can be used to focus the electromagnetic radiation 410 to a small eye tissue area. The imaging lens 430 can be a spherical convex lens, an aspheric lens, an objective lens, a theta lens, or a cylindrical lens for line focusing.

[0039] To scan the axial position of the focal spot in the ocular tissue, the imaging lens 430 can be mounted on a translation stage 434. Alternatively or additionally, a variable element can be used that can vary the divergence of the probe radiation. To scan the lateral position of the focal spot, a one or two axis beam scanner 440 can be used. This example scanner 440 can include a galvanometer mounted mirror, a MEMS mirror, a translation stage, a spatial light modulator, or the like.

[0040] Acousto-optical interactions in tissue can result in light / radiation scattering, which generates at least one second electromagnetic radiation. Several light / radiation scattering mechanisms are known in the art, including Rayleigh and Mie scattering, Raman scattering, and Brillouin scattering. Biological tissue aids in these scattering mechanisms, but Brillouin scattering is directly related to acoustic waves in the medium. A portion of such one or more second electromagnetic radiations can be collected by imaging lens 430.

[0041] The example system of Figure 4 may use a beam splitter 442 that reflects and transmits the first and second electromagnetic radiation. The beam splitter 442 may have, for example, an equal 50:50 split ratio or an unequal split ratio to optimize efficiency of signal generation and collection. The beam splitter 442 may be a neutral splitter with a wide spectral bandwidth, or a dichroic splitter based on multi-layer coating, interference, or diffraction. A portion of the second electromagnetic radiation 444 may be sent to the second array 406, which may be configured to receive at least a portion 444 of the one or more second electromagnetic radiations 444 described above.

[0042] Instead of the "desktop" configuration shown in Figures 3 and 4, the human interface can use a fiber optic probe in the form of an endoscope or a catheter, as shown in the three configurations in Figure 5. The pen-type endoscopic probe 560 of Figure 5A includes an optical fiber 561 held by a holder 562 and a focus knob 564 configured to position a guide rail 563 for proper generation of electromagnetic radiation from a lens 565 of the probe 560. The pen-type endoscopic probe 570 of Figure 5B has a similar configuration to that of 560, but has a fixed focal length. The endoscopic probe 570 includes an optical fiber 571 and a guide rail 573, but also includes a voice coil 575 and a wire 576. The catheter probe 580 of Figure 5C includes an optical fiber 581 connected to a rotary coupling 582, which is connected to a catheter 583. Catheter 583 is comprised of a sheath 584, a shaft coil 585 that encases optical fiber 583, a GRIN lens 586, and a mirror prism 587. Other equivalent components or arrangements may also be used.

[0043] The pen endoscope shown in Fig. 5A and Fig. 5B can be used to non-invasively examine the sclera through the conjunctiva or to probe the lens tissue behind the pupil during surgery. The catheter fiber optic probe shown in Fig. 5C can be used for intravascular measurements of blood vessel walls or for endoscopic examination of the gastrointestinal and airways. A fiber optic probe integrated into a needle can be used to identify cancerous areas with large tissue masses. The fiber probe can have two separate optical fibers at the input and output ports, similar to Fig. 3A with two separate fibers. More preferably, the endoscope and catheter can have one fiber for the two ports, the input and output ports. Fig. 5 shows three schematic diagrams of different embodiments of such optical probes. The probe can be directly coupled to a laser source system and a spectrometer by optical fibers. The probe can have two separate input and output fibers. However, it is possible and preferred to use a single fiber to achieve the coupling to the laser source and spectrometer via an optical circulator, fiber optic (2x2) splitter, or similar component.

[0044] In a single fiber configuration, the optical fiber itself will have natural Brillouin scattering, and the Brillouin backscattered light may be combined with the Brillouin signal from the sample. Since the length of the optical fiber can be on the order of several meters, the magnitude of the Brillouin light coming from the optical fiber may be several to many orders of magnitude larger than the magnitude of the Brillouin light signal from the sample. For a silica optical fiber, the Brillouin frequency shift is approximately 21-22 GHz at an optical wavelength of 780 nm. If this spectrum overlaps with the Brillouin spectrum of tissue, which is typically 5-8 GHz, it becomes difficult to accurately determine the Brillouin shift of the tissue. As a result, the system may have a spectral resolution configuration that removes the Brillouin light coming from the fiber or separates the two Brillouin spectra from each other. In one embodiment, the VIPA etalon has an FSR equal to the Brillouin shift of the optical fiber (e.g., 21-22 GHz) or half or 1.5 times that, such that the Brillouin spectrum coming from the fiber due to reflection or elastic scattering of the laser light will overlap with the background spectrum and be separated from the Brillouin signal spectrum from the sample.

[0045] Additionally or alternatively, a non-reciprocal polarization rotator can be used at the distal end of the lead fiber prior to the sample to make the polarization states of the Brillouin scattered light emerging from the fiber orthogonal to the polarization states of the Brillouin scattered light emerging from the sample, and a polarizing beam splitter can then be used at the proximal end of the lead fiber to send only the Brillouin signal emerging from the sample to the spectrometer.

[0046] <Spectrometer>

[0047] The spectrometer system 106 may be of any known type suitable to function with a selected laser source system and human interface. The spectrometer system 106 may be configured to receive a portion of the scattered electromagnetic radiation to provide information related to the biomechanical properties of the animal tissue.

[0048] 6 shows one embodiment of a spectrometer system 606 that uses a rubidium vapor absorption filter to attenuate the laser frequency components while transmitting the Brillouin scattered light with low insertion loss. The system includes a Rb cell 610 with a heater 612, a cylindrical lens (CL), a mirror (M), a VIPA etalon 614, a mask 616, and a charge-coupled device (CCD) 618 as a detector. The spectrometer system 606 can also use a calibration device, which can be a temperature stabilized calibrant or an electro-optical frequency modulator that generates sidebands for high-precision calibration of the spectrometer.

[0049] Previous systems used two VIPA etalons in two stages. The optical insertion loss of the second stage is typically 5-6 dB higher than the first stage by 3-4 dB. The Rb notch filter provides sufficient rejection to allow the use of only one VIPA stage. Typical single stage attenuation is 30-40 dB, so the total attenuation of the rejection filter and VIPA is >90 dB. The single stage configuration reduces size, complexity, cost, and importantly, optical loss. It also allows line scanning or simultaneous detection and readout from a fiber bundle.

[0050] In the detection path, a high vapor pressure Rb absorption filter can be used to remove Fresnel reflected and Rayleigh scattered light with high attenuation from the light collected from the human interface. The Rb notch filter can remove these spurious elastic components with the same frequency as the laser line from the light before it enters a spectrometer, such as the VIPA etalon spectrometer shown. Although a multi-pass FP etalon-based filter can provide high attenuation of around 40 dB, its mechanical stability is low and it is sensitive to temperature. As shown by the attenuation spectrum in Figure 7, experimental results show that a high pressure Rb vapor cell filter can provide a much higher single-pass rejection of more than 60 dB or even 80 dB when the Rb gas is heated at 65 °C in an insulated furnace. The insertion loss of the vapor absorption filter can be less than 1 dB. The high pressure Rb vapor cell filter can be compatible by itself with a Rb laser source frequency-locked to the same transition line of the Rb atom.

[0051] The spectrometer system 606 in FIG. 6 can be equipped with a low-cost thermoelectrically cooled CCD camera instead of the expensive EM-CCD camera. For example, a commercially available camera developed for astronomy applications has a high quantum efficiency of 65% at 780 nm and a low read noise of about 3-6 electrons. To obtain a frequency measurement sensitivity of about 10 MHz, a total of about 1,000 signal electrons are required across several pixels. When the number of Brillouin signal photons exceeds 56 (36 electrons) per pixel, the shot noise becomes larger than the read noise. The penalized signal-to-noise ratio (SNR) due to the read noise and the slightly lower quantum efficiency is less than 3 dB. Experimental results show that the signal increase of about 5-6 dB achieved by removing the second stage of the VIPA is sufficient to offset the read noise impairment (less than 3 dB) and the insertion loss of the rejection filter (1 dB) to not reduce, and in some cases improve, the overall detection sensitivity of the system.

[0052] Previous systems used VIPA etalons made from fused silica, whose refractive index is temperature dependent (10~5 / °C). The use of silica etalons can cause the diffraction pattern at the CCD to shift by 3.8 GHz / °C depending on the ambient temperature, and such temperature sensitivity is sufficiently slow (e.g., less than 10 MHz / min or 2.6×10) that the shift can be corrected by calibration using reference materials at the human interface. -3 This may only be acceptable in a temperature-regulated laboratory environment where the thermal expansion coefficient tends to be about 5×10° C. / min. However, this temperature sensitivity needs to be improved for use in more general environments. To solve this problem, the spectrometer 606 is -9 A VIPA etalon made of ultra-low expansion (ULE) glass such as Zerodur®, which has a thermal sensitivity of 1 / °C, is used. Such a ULE VIPA etalon will have a temperature sensitivity 2000 times lower than a silica VIPA etalon, which results in no calibration error for temperature gradients up to 5.2°C / min. Additionally or alternatively, a small heater and TEC can be used to stabilize the temperature of either the ULE etalon or the silica etalon.

[0053] <Computer System>

[0054] The computer system 108 may be electrically or wirelessly connected to the laser source system 102, the human interface 104, the spectrometer system 106, and / or other additional components of the Brillouin imaging system. The computer system 108 may be used to provide feed-forward or feedback control during use of the Brillouin imaging system 100. In this manner, the computer system 108 may affect the output of the laser source system and / or modify human interface parameters, such as the angle of incidence, in real time.

[0055] 8 is a simplified block diagram of a computer system 808 that may be used in the Brillouin imaging system of the present disclosure. The computer system 808 typically includes at least one processor 852, which may communicate with a number of peripheral devices via a bus subsystem 854. These peripheral devices may include a storage subsystem 856 having a memory subsystem 858 and a file storage subsystem 860, a user interface input device 862, a user interface output device 864, and a network interface subsystem 866. The network interface subsystem 866 provides an interface to an external network 868 and / or other devices, such as a tissue assessment system.

[0056] The user interface input devices 862 may include keyboards, pointing devices such as mice, trackballs, touch pads, or graphics tablets, scanners, foot pedals, joysticks, touch screens integrated into displays, audio input devices such as voice recognition systems, microphones, and other types of input devices. The user input devices 862 are often used to download computer executable code from a tangible storage medium that implements one of the methods of the present invention. In general, the term "input device" is intended to include a variety of conventional and proprietary devices and techniques for inputting information into the computer system 808.

[0057] The user interface output devices 864 may include a display subsystem, a printer, a fax machine, or a non-visual display such as, for example, an audio output device. The display subsystem may include a cathode ray tube (CRT), a flat panel device such as, for example, a liquid crystal display (LCD), or a projection device. The display subsystem may also provide a non-visual display such as, for example, via an audio output device. The term "output device" as used generally is intended to include a variety of conventional and proprietary devices and techniques for outputting information from the computer system 808 to a user.

[0058] Storage subsystem 856 may store the basic programming and data structures that provide the functionality of various aspects of the present disclosure. For example, databases and modules that embody the functionality of the inventive methods described herein may be stored in storage subsystem 856. Such software modules are typically executed by processor 852. In a distributed environment, software modules may be stored in multiple computer systems and executed by processors of these multiple computer systems. Storage subsystem 856 typically includes a memory subsystem 858 and a file storage subsystem 860.

[0059] The memory subsystem 858 typically comprises multiple memories, including a main random access memory (RAM) 870 for storing instructions and data during program execution, and a read-only memory (ROM) 872 in which persistent instructions are stored. The file storage subsystem 860 provides persistent (non-volatile) storage of program and data files and may include tangible storage media that may optionally implement tissue assessment data. The file storage subsystem 860 may include hard disk drives, floppy disk drives with associated removable media, compact disk read-only memory (CD-ROM) drives, optical drives, DVDs, CD-Rs, CD-RWs, solid state removable memories, and / or other removable media cartridges or disks. One or more of the above drives may be located on other remotely connected computers at other locations coupled to the computer system 808. Modules embodying the functionality of the methods disclosed herein may be stored by the file storage subsystem 860.

[0060] Bus subsystem 854 provides a mechanism that allows the various components and subsystems of computer system 808 to communicate with each other as intended. The various subsystems and components of computer system 808 need not be physically co-located, but may be distributed across multiple different locations in a distributed network. Although bus subsystem 854 is illustrated generally as a single bus, alternative embodiments of the bus subsystem may use multiple buses.

[0061] The computer system 808 itself can be of a wide variety of types, including a personal computer, a portable computer, a workstation, a computer terminal, a network computer, a control system in a wavefront measurement system or a laser surgery system, a mainframe, or any other data processing system. Because the nature of computers and networks is constantly changing, the description of the computer system 808 shown in Figure 8 is intended only as a specific example for purposes of illustrating one aspect of the present disclosure. Many other configurations of computer system 808 are possible, including those with more or fewer components than the computer system shown in Figure 8.

[0062] <Differential method>

[0063] The subtraction techniques described herein can be used in conjunction with the improved Brillouin imaging systems described above or with traditional systems to better assess a patient's ocular tissue.

[0064] It has been discovered that many confounding factors affect the biomechanical properties of tissues globally, yet biomechanical information of interest can appear locally: for example, temperature affects the entire cornea rather uniformly, whereas pathological changes resulting from keratoconus can occur in a heterogeneous or spatially dependent manner.

[0065] A patient can be evaluated using a difference metric based on changes in elasticity in the cornea (e.g., by considering the difference in elasticity between the thinnest point of the cornea and the center point of the cornea) or a difference metric based on the difference in corneal elasticity between the patient's two eyes. The thinnest point of the cornea can be identified by a variety of techniques, including corneal topography approaches that identify the tightest curvature of the cornea. Pachymetry can also be used to identify the thinnest point of the cornea. Once such a difference elasticity value is obtained, it can be used in place of an absolute elasticity value, which can vary widely and overlap between normal populations and patient populations with keratoconus or other injuries. Keratoconus is typically a bilateral disease with differences between the eyes in the time of onset. The example difference metrics have desirable sensitivity and specificity in distinguishing between normal and abnormal corneas.

[0066] In one aspect, the present disclosure provides a method for measuring the difference in biomechanical properties between two ocular tissue regions and generating a metric based on the difference. By accounting for this difference, the uniform contribution of confounding factors can be reduced or offset, thereby reducing or eliminating errors in the interpretation of the measurement results. As a result, the system and method can use a metric that improves the detection of desired biomechanical information of tissues that may be compromised by confounding factors.

[0067] FIG. 9 illustrates a process flowchart 900 of a differential technique in which a first biomechanical value of a first ocular tissue region is determined (902) using Brillouin spectroscopy, a second biomechanical value of a second ocular tissue region is determined (904), and the first biomechanical value is compared (906) to evaluate the patient's ocular tissue.

[0068] Binocular evaluation Symmetry between healthy eyes of the same individual is well known in the literature, including the symmetry of the distribution of biomechanical properties of the human cornea. Investigations using X-ray scattering techniques have shown that the stroma, which represents approximately 90% of the corneal thickness, contains highly anisotropic arrangements of collagen fibers that are preferentially aligned in a pattern. The structure is symmetric between the eyes. This results in a symmetric distribution of biomechanical properties in the corneas of both eyes of the same individual.

[0069] In a binocular approach, a method for evaluating a patient's ocular tissue may include determining a first biomechanical value of a first ocular tissue region located in the patient's eye, determining a second biomechanical value of a second ocular tissue region located in the patient's other eye, and comparing the first and second biomechanical values ​​to evaluate the patient's ocular tissue. The biomechanical value may be an elasticity value. The first and second tissue regions may each be selected from the group consisting of corneal tissue, scleral tissue, and lens tissue.

[0070] Alternatively, example methods can include obtaining biomechanical values ​​from both eyes and then generating a metric related to the difference in the biomechanical values ​​between the left and right eyes. The second biomechanical value can be obtained from Brillouin spectroscopy or Brillouin microscopy. Alternatively, the second biomechanical value can be obtained by air-puff optical imaging, such as Scheimpflug topography or optical coherence tomography, and the biomechanical value can be related to the air-puff induced deformation of the cornea.

[0071] Monocular evaluation In a monocular approach, a method for evaluating an ocular tissue of a patient may include determining a first biomechanical value of a first ocular tissue region located on the patient's eye, determining a second biomechanical value of a second ocular tissue region located on the patient's eye, and comparing the first biomechanical value to the second biomechanical value to evaluate the ocular tissue of the patient. The biomechanical value may be an elasticity value. The first tissue region and the second tissue region may each be selected from the group consisting of corneal tissue, scleral tissue, and lens tissue.

[0072] In all studied keratoconus patients with stages 1 to 4, the Brillouin shift values ​​measured in the cone region were significantly lower than those in the peripheral region (3 mm away from the optical axis) in the same eyes (P<0.05). In contrast, the Brillouin shift values ​​in the cone region of keratoconus patients were not significantly and distinguishably different from those measured in the central cornea of ​​normal subjects (p>0.05), which may be due to a relatively large intersubject variability.

[0073] The above success stories based on a comparison of the left and right eyes of each subject, as well as a comparison of regional differences on the same cornea, demonstrate the utility of "self-calibration," providing a calibration standard from other regions of the same eye or the other eye of the subject.

[0074] [example] The following examples are provided to illustrate and further illustrate certain embodiments and aspects of the present disclosure and are not to be construed as limiting the scope of the present disclosure.

[0075] Example 1 A study was conducted to evaluate the effectiveness of the systems and methods described herein and the perturbing factors that necessitate these solutions.

[0076] Corneal hydration is a dynamic equilibrium that is maintained or influenced by multiple confounding factors, including binocular variations due to environmental and physiological changes, as shown in Figure 10. The normal water content of the cornea can be kept constant by a balance between factors that draw water into the cornea (e.g., tumescent pressure and intraocular pressure), factors that prevent water flow in the cornea (e.g., the epithelial barrier), and factors that pull water out of the cornea (e.g., the endothelial pump).

[0077] In clinical practice, changes in corneal hydration can be indirectly estimated using central corneal thickness (CCT) because corneal expansion is perpendicular to the tangent direction. An increase in CCT indicates increased water content and reduced elasticity, which reduces the Brillouin frequency shift. In this study, we measured the daily changes in CCT and the corresponding central corneal Brillouin frequency shift and confirmed the trend.

[0078] Another confounding factor in Brillouin spectroscopy is temperature. Figure 11 shows the experimentally measured Brillouin frequency shift at 780 nm in pure water in the temperature range of 15-40°C. The dotted line represents the raw data, and the dashed line represents the fitted quadratic polynomial. The temperature dependence of corneal tissue is similar to that of water, about 5 MHz / °C. Corneal temperature is known to vary from person to person and has some correlation with age and daily physiological conditions. At room temperature, the corneal temperature is several degrees Celsius to many degrees Celsius lower than body temperature. This is due to heat transfer to the surrounding air. Thus, the ambient temperature also affects the corneal temperature. Thus, the Brillouin imaging system disclosed herein can be equipped with a temperature measuring device, such as an infrared detector, to measure the corneal temperature and correct for the temperature dependence. A thermometer can also be equipped to measure the ambient temperature, in order to estimate the corneal temperature using simple thermal modeling of the eye.

[0079] Diurnal variations in central corneal Brillouin and corneal hydration (CCT) measurements were evaluated in healthy subjects. Figure 12A shows the diurnal variations in Brillouin frequency shift and CCT measurements obtained from a healthy subject. The left column shows the raw data acquired over the entire day, starting 1 hour after waking. The right column shows the interocular differences in Brillouin shift and CCT measurements. The results show that the interocular differences in both measurements are very small and within the range of the system error (<±10 MHz and <10 μm).

[0080] In another study, we measured the central corneal Brillouin frequency shift OD-OS difference in 37 patients. The results from both corneas showed a very narrow range, with the difference between the two eyes falling within a distribution with a standard deviation of <10 MHz, as shown in Figure 12B.

[0081] Example 2 A study was conducted to evaluate the effectiveness of the systems and methods described herein and the effects on diseases requiring these solutions.

[0082] Keratoconus, a corneal degenerative disease, is an example of an eye disease that can be evaluated using the disclosed systems and methods. The onset of keratoconus is often unilateral. For example, keratoconus may begin in one eye before the other eye begins to develop abnormal changes. Brillouin frequency shifts were measured in both eyes of subjects with one eye classified as early and mild stage 1 keratoconus according to standard pachymetry readings and the Amsler-Krumeich classification and the other eye normal. Brillouin biomechanical values ​​measured in the central superior region (0.8-1 mm below the optical center) were statistically significantly different between the two eyes (p<0.0001, 4 subjects). Brillouin values ​​measured in the thinnest region of the cornea (0.5-1 mm diameter) were also significantly different between the two eyes. For each subject, the Brillouin shift was lower in the stage 1 eye than in the normal eye. In contrast, the difference between the two eyes of normal healthy patients (n=20) was lower than the measurement precision (±10 MHz).This example shows that biomechanical differences between the left and right eyes can be used to detect early keratoconus.

[0083] In a separate study, Brillouin frequency shifts were measured in both eyes of subjects classified as having early, mild stage 1 keratoconus according to standard pachymetry readings and the Amsler-Krumeich classification. Figure 13 (left set) shows a typical example obtained from a stage 1 patient. Brillouin biomechanical values ​​measured in the thinnest (cone) area of ​​the cornea (R<1mm from the thinnest point of the cornea) were significantly different between the two eyes (n=4, p<0.0001). Corneal thickness and posterior elevation maps were obtained using a Scheimpflug corneal topography system along with Brillouin elasticity maps for a patient (38.5±5.73y / d, 2 males) diagnosed with early stage keratoconus (stage I) in both eyes. The dashed circle indicates the cone area R<1mm around the thinnest point.

[0084] In contrast, Figure 13 (right set) shows that the difference between the eyes of normal healthy patients (n=37) is less than the measurement precision (±10 MHz). Absolute OD-OS difference of central Brillouin measurements in normal corneas (left dotted line) and eight stage I keratoconus of four patients (right dotted line). The yellow dotted line shows the central difference (R<1mm from pupil center). The right dotted line shows the minimum Brillouin difference between the cones of both eyes ( *** p<0.001). This case demonstrates that the biomechanical differences between the left and right eyes can be used to detect early keratoconus. Thus, Figure 13 demonstrates the diagnostic validity of Brillouin measurement parameters for keratoconus.

[0085] The above successes based on comparison of the left and right eyes of each subject and comparison of regional differences in the same cornea demonstrate the utility of "self-calibration" to provide a calibration standard with other regions of the same eye or the other eye of the subject. In patients with stage III and IV keratoconus, localized reductions in elasticity measured using Brillouin spectroscopy were observed. Regional heterogeneity was measured in patients with early stage keratoconus, and the results are shown in Figure 14, which shows a representative Brillouin image of a cornea with stage I keratoconus. Rows 2-4 are topography images obtained with a Scheimpflug principle imaging system commonly used in clinical practice, showing thickness (μm), corneal curvature measurement (or sagittal curvature, in diopters), and posterior corneal surface elevation (μm).

[0086] When analyzing data from patients with stage I-IV keratoconus, the Brillouin shift values ​​measured in the cone region were significantly (P<0.001, paired t-test) lower than those in the peripheral region (R>3 mm from the optical axis) in the same eye, and the regional differences increased with disease severity, as shown in Figures 15A and 15B. In contrast, no differences were found when comparing Brillouin shift values ​​across subjects. Brillouin values ​​in the cone region of keratoconus patients were not distinguishably different (p>0.05) from those measured in the central cornea of ​​normal subjects. This low sensitivity is mainly due to the relatively large inter-subject variability. Figure 15A shows the regional differences in Brillouin frequency shift measurements in keratoconus. As shown in FIG. 15B, the central region can be defined as less than 1 mm from the pupil center, and the cone region in keratoconic corneas can be defined as the region less than 1 mm from the thinnest point as defined by Scheimpflug corneal topography. FIG. 15B shows regional differences in Brillouin measurements in stages I-IV keratoconus. Statistical significance was confirmed by two-sided, unpaired student tests ( ** p<0.01, *** p<0.001).

[0087] Some Fuchs dystrophies are associated with a loss of endothelial cell function in water transport. Corneal thickness recovery in response to induced hydration changes has been proposed as a test of endothelial function. Brillouin microscopy can be used to measure abnormal hydration changes in patients with Fuchs dystrophy to aid in monitoring the progression of the disease. For example, metrics such as minimum Brillouin shift and the difference between minimum (or central) and peripheral Brillouin values ​​can be used to measure distention to diagnose and monitor treatment of Fuchs dystrophy.

[0088] Some dry eye syndromes are due to chronic problems with the tear film that do not adequately lubricate or hydrate the corneal surface. It is believed that corneal tissue in dry eye is less hydrated and therefore has a higher Brillouin shift than normal cornea. Thus, the Brillouin frequency shift can serve as a useful index in diagnosing and monitoring the treatment of dry eye patients.

[0089] Some collagen-rich tissues, such as the cornea, skin, and muscle, are highly anisotropic, and their biomechanical and optical properties are direction-dependent. It has been found that when the light beam is oriented at an angle to the corneal surface, the Brillouin shift of the cornea is 50-100 MHz higher than when the beam is incident on the cornea perpendicular to the corneal surface.

[0090] In the corneal stroma, collagen fibrils are organized in layers that have different orientations in the xy plane. The Brillouin frequency, f, measured as a function of the angle θ with respect to the normal to the corneal plane, is given by f≒f 0 +Δf sin θ It can be expressed as:

[0091] where f 0 is measured perpendicular to the cornea, and Δf represents the axial asymmetry of the tissue. Δf can range from 0 to 1 GHz depending on the material. A typical cornea has a Δf of about 200 to 400 MHz, measured with an optical wavelength of 780 nm.

[0092] This asymmetry or frequency difference can be measured by varying the angle of the probe beam axis with respect to the corneal surface normal. For example, the two reference axis values ​​can be determined from the following two measurements:

number

[0093] where f nrepresents the Brillouin shift along the axis perpendicular to the corneal surface, and f p represents the Brillouin shift along an axis parallel to the corneal surface, and f1 and f2 represent the inclination angle θ 1 and θ 2 are the measured Brillouin shifts, respectively.

[0094] Most of the moisture or hydration effects are spatially isotropic and should be independent of the Brillouin probe beam angle. Thus, hydration does not significantly affect Δf. Therefore, the axial asymmetry metric represents the mechanical properties of the solid components, especially the collagen fiber structure. Changes in collagen fibril thickness, density, and crosslinks can appear as an axial asymmetry metric.

[0095] Since two or more measurements of the same cornea are taken, the contribution of confounding homogeneous factors can be cancelled out, and thus the anisotropic factor, as represented by the angle difference, can be a sensitive metric for monitoring corneal health and diagnosing corneal disease.

[0096] Myopia is associated with deformation and stretching of the eye, with the sclera weakening and becoming more stretchable, making it unable to withstand the expansive forces of intraocular pressure. The sclera is a tough outer shell that protects and supports the more delicate intraocular structures. Investigations have identified ultrastructural changes, tissue loss, and biomechanical remodeling in the sclera of myopic eyes. In myopic eyes, the scleral thickness and cross-sectional area at the equator, the midpoint between the equator and the posterior pole, the optic disc border, and the posterior pole decrease significantly with the axial length of the eye. Since scleral remodeling is triggered in situ at the onset of myopia, the localized decrease in elasticity in that region can be used to predict pathological myopia, which can be quantified using the Brillouin imaging systems and methods described herein.

[0097] Cross-linking is a medical procedure that can be used to treat corneal ectasia and abnormal eye growth (e.g., causing myopia) by stiffening the cornea or sclera. Cross-linking can be achieved by instilling dyes, such as riboflavin and rose bengal, into the eye tissue and applying light. An example of a related cross-linking technique is described in U.S. Patent Application Publication No. 2016 / 0151202, the contents of which are incorporated herein by reference. The Brillouin frequency shift can be measured in different regions where changes in the degree of cross-linking can be caused, and the difference in the Brillouin value between each region can provide information to confirm whether the treatment was performed properly or effectively. For example, the cone region of a keratoconus patient can be irradiated with a higher intensity of UVA light, while the surrounding region can be irradiated with a lower intensity of UVA light. The difference between the regions can be calculated, especially in comparison with the difference between the regions measured before the cross-linking procedure. The difference is expected to be increased after cross-linking. Otherwise, a lack of change or a smaller than expected change suggests that the treatment may not have been effective. Scleral cross-linking can measure the difference in Brillouin frequency shift between the equatorial and posterior sclera before and after cross-linking. In this regard, the change in the difference from region to region can also be used as a metric to assess the effectiveness of cross-linking.

[0098] Another example is myopic anisometropia, where the two eyes of the same individual with the same biological background and exposed to the same main environmental factors develop significantly different refractive errors. Anisometropia can be characterized by intraocular asymmetry. Research has suggested that intraocular asymmetry is related to the magnitude or rate of growth of the posterior segment of the eye. This suggests that the structural and biomechanical development of the two eyes is different, and therefore characterizing the differences in the same region of the sclera of both eyes, e.g., the equatorial region, may help identify anisometropia early for early intervention.

[0099] 16 illustrates multiple views of a Brillouin imaging system 100 according to an aspect of the present disclosure, which may include an optical coherence tomography (OCT) modality, a Brillouin imaging modality, a Raman imaging modality, a laser speckle imaging modality, a multiphoton imaging modality, a photoacoustic imaging modality, a confocal microscopy imaging modality, a fluorescence imaging modality, a Pentacam® imaging modality, an ultrasound imaging modality, and assemblies combining or including one or more of the above imaging modalities. In this regard, examples of tissue evaluation or tissue assessment techniques that can be used with the tissue evaluation system 100 and methods include U.S. Patent Nos. 7,898,656, 8,115,919, 9,777,053, U.S. Patent Application Publication Nos. 2012 / 0302862, and 2016 / 0151202, the contents of which are incorporated herein by reference.

[0100] As shown in FIG. 16, the tissue evaluation system 100 can be configured to obtain a first biomechanical value of a tissue of a patient's eye 1612. The system 100 can also be configured to obtain a second biomechanical value of a fellow eye 1614 of the patient. Additionally, the system 100 can be configured to compare the first biomechanical value to a second biomechanical value to evaluate the tissue of the eye. As shown in the figure, the eye 1612 is a keratoconic eye and the fellow eye 1614 is a normal or healthy eye. In some cases, the first biomechanical value is an elasticity value of a tissue of the eye and the second biomechanical value is an elasticity value of a corresponding tissue of the fellow eye. In some cases, the tissue is corneal tissue 1616. In some cases, the tissue is scleral tissue 1617. In some cases, the tissue is lens tissue 1618. In some cases, the first biomechanical value is obtained by Brillouin spectroscopy. In some cases, the first biomechanical value is obtained by Brillouin microscopy. In some cases, the first biomechanical value is obtained by an air-puff optical imaging technique. The air-puff optical imaging technique can be, for example, a Scheimpflug topography technique or an optical coherence tomography technique. In some cases, the first biomechanical value of the patient's ocular tissue can be a biomechanical value obtained at a central inferior region of the cornea of ​​the eye, and the second biomechanical value of the corresponding tissue of the patient's other eye can be a biomechanical value obtained at a central inferior region of the cornea of ​​the other eye. In some cases, the first biomechanical value of the patient's ocular tissue can be a biomechanical value obtained at a thinnest region of the cornea of ​​the eye, and the second biomechanical value of the corresponding tissue of the patient's other eye can be a biomechanical value obtained at a thinnest region of the cornea of ​​the other eye. Although FIG. 16 illustrates a binocular evaluation technique, embodiments of the invention include monocular tissue evaluation techniques as described elsewhere herein.

[0101] In example embodiments of the present invention, systems and methods can be used to perform in vivo Brillouin microscopy on ocular tissue, which can be valuable in both characterizing the biomechanics of the eye in diagnosing and treating ocular problems, and in the development of new drugs or treatments.

[0102] There are four anatomical regions in the eye. For example, the cornea is a thin (e.g., less than 1 mm) tissue composed of multiple layers with different mechanical strengths. Aqueous humor is a liquid with properties similar to water that fills the anterior chamber of the eye. The crystalline lens is a biconvex sphere composed of multiple layers with different refractive indices, densities, and stiffnesses. The vitreous is a viscous, clear liquid that fills the posterior chamber of the eye.

[0103] Brillouin light scattering in tissue or any other medium usually occurs due to the interaction of incident light with acoustic waves in the material. For example, a probe light with frequency ν and wavelength λ can be used, which can be fed to the sample. In the natural Brillouin process, acoustic waves or acoustic phonons exist naturally due to thermal fluctuations. Such fluctuations propagate in the medium in the form of acoustic waves. The acoustic waves can cause a periodic modulation of the refractive index. Brillouin scattering can be caused by at least one or many acoustic waves or acoustic phonons, which form a phase-matched index modulation.

[0104] All patent related submissions, scientific journals, books, papers, and other publications and materials mentioned herein are hereby incorporated by reference for all purposes. Various modifications are possible within the scope of the present invention. Various parameters, variables, factors, etc. may be incorporated into the steps of the exemplary method or modules of the exemplary system. Although certain embodiments have been described in detail by way of example for ease of understanding, various adjustments, modifications, and improvements will be apparent to those skilled in the art.

[0105] All system and / or apparatus configurations described herein are applicable to the methods described herein and vice versa. Each of the calculations described herein can be performed using a computer or other processor with hardware, software and / or firmware. The various steps of the methods can be performed by modules, which can comprise any of a wide variety of digital and / or analog data processing hardware and / or software configured to perform each step of the methods described herein. The modules comprise data processing hardware suitable for performing one or more of the steps by providing appropriate machine programming code associated with that step or steps, where modules for two or more steps (or parts of two or more steps) are integrated on one processor board or separated on separate processor boards in any of a wide variety of embedded and / or distributed processing architectures. These methods and systems often employ a tangible medium having machine readable code embodied therein having instructions for performing each step of the methods described herein. Suitable tangible media may include memory (including volatile and / or non-volatile memory), or storage media (e.g., magnetic recording on floppy disks, hard disks, or tapes, e.g., recording on CDs, CD-R / Ws, CD-ROMs, or DVDs, or any other digital or analog storage media), etc. Although the above example embodiments have been described in some detail for illustrative purposes, those skilled in the art may make various improvements, adaptations, and modifications.

[0106] The disclosed methods and devices may be provided in one or more kits for use. Such kits may include a system or device for evaluating patient tissue and instructions for use. Optionally, such kits may further include any of the other system components described in connection with the present invention, and any other materials or items related to the present invention. The instructions for use may describe any of the methods described above.

[0107] While the present invention has been described in terms of one or more preferred embodiments, it is apparent that many equivalents, alternatives, variations, and modifications other than those expressly described are possible and are within the scope of the invention.

Claims

1. 1. A Brillouin spectroscopy system for evaluating tissue in an ocular tissue region of a patient, comprising: a tunable laser source configured to generate a first electromagnetic radiation having an electromagnetic spectrum; a vapor cell-based reference configured to acquire a portion of the first electromagnetic radiation and generate an error signal that includes information regarding a deviation of the electromagnetic spectrum from a target electromagnetic spectrum; The laser light source system includes: The vapor cell type reference part is a vapor cell filter configured to receive the portion of the first electromagnetic radiation and selectively transmit the portion of the first electromagnetic radiation based on the electromagnetic spectrum; a detector configured to receive the transmitted portion of the first electromagnetic radiation and generate the error signal used to control the tunable laser light source; Equipped with The Brillouin spectroscopy system further comprises a human interface configured to deliver the first electromagnetic radiation to the ocular tissue region of the patient; the first electromagnetic radiation interacting with at least one acoustic wave endogenous to the ocular tissue region to generate at least one second electromagnetic radiation based on the at least one acoustic wave; The Brillouin spectroscopy system further comprises a spectrometer system; The spectrometer system comprises: a rubidium vapor absorbing filter that receives the first electromagnetic radiation and the second electromagnetic radiation and transmits the second electromagnetic radiation and attenuates the first electromagnetic radiation; a detector configured to receive a portion of the second electromagnetic radiation from the rubidium vapor absorbing filter without receiving the first electromagnetic radiation from the rubidium vapor absorbing filter, and to provide information related to a biomechanical property of the ocular tissue region; A Brillouin spectroscopic analysis system comprising:

2. The target electromagnetic spectrum is the wavelength of the absorption line of an atomic species; 2. The Brillouin spectroscopy system of claim 1.

3. The target electromagnetic spectrum is the wavelength of rubidium absorption lines.

3. The Brillouin spectroscopy system of claim 2.

4. The target electromagnetic spectrum is at a wavelength of 780 nm; 4. The Brillouin spectroscopy system of claim 3.

5. and a spectral clean-up filter configured to generate a second error signal that includes information about spectral noise in the first electromagnetic radiation.

2. The Brillouin spectroscopy system of claim 1.

6. the human interface comprises a fiber optic probe; 2. The Brillouin spectroscopy system of claim 1.

7. the fiber optic probe is a catheter; 7. The Brillouin spectroscopy system of claim 6.

8. The fiber optic probe is a pen-type endoscope.

7. The Brillouin spectroscopy system of claim 6.

9. the Brillouin spectroscopy system further comprises a computer system in communication with the human interface and the spectrometer system; The computer system is configured to obtain a first biomechanical value of a first ocular tissue region of a patient, obtain a second biomechanical value of a second ocular tissue region of the patient, and identify a medical condition of the tissue of the patient's eye by comparing the first biomechanical value to the second biomechanical value.

2. The Brillouin spectroscopy system of claim 1.

10. The computer system is further configured to assess axial asymmetry between the first ocular tissue region and the second ocular tissue region to identify the medical condition.

10. The Brillouin spectroscopy system of claim 9.

11. The computer system is further programmed to determine a Brillouin frequency f measured in dependence on an angle θ relative to each normal at the first eye tissue region and the second eye tissue region.

11. The Brillouin spectroscopy system of claim 10.

12. The Brillouin frequency f is f≒f ０ +Δfsinθ where f ０ is a value measured perpendicular to the first eye tissue region or the second eye tissue region, and Δf is the axial asymmetry between the first eye tissue region and the second eye tissue region.

12. The Brillouin spectroscopy system of claim 11.

13. 1. A Brillouin spectroscopy system for producing a peak frequency stabilized and spontaneous emission noise filtered laser, comprising: a tunable laser source configured to generate a first electromagnetic radiation having an electromagnetic spectrum; a vapor cell-based reference configured to acquire a polarized portion of the first electromagnetic radiation and generate an error signal that includes information regarding a deviation of the electromagnetic spectrum from a target electromagnetic spectrum; Equipped with The vapor cell type reference part is a first polarizer configured to receive the portion of the first electromagnetic radiation and change the polarity of the portion; a vapor cell configured to receive the portion of the polarized form of the first electromagnetic radiation from the first polarizer; a second polarizer configured to receive the portion of the polarized form of the first electromagnetic radiation from the vapor cell and change the polarity of the portion of the first electromagnetic radiation; a detector configured to receive the portion of the polarized form of the first electromagnetic radiation from the second polarizer and generate an error signal; a spectral clean-up filter configured to generate a second error signal that contains information about spectral noise in the first electromagnetic radiation; Equipped with The spectral clean-up filter comprises: filtering at least a portion of the spectral noise; or It has two tandem Fabry-Perot (FP) cavities, each consisting of a pair of concave mirrors. The Brillouin spectroscopy system further comprises a human interface configured to deliver the first electromagnetic radiation to an ocular tissue region of a patient; the first electromagnetic radiation interacting with at least one acoustic wave endogenous to the ocular tissue region to generate at least one second electromagnetic radiation based on the at least one acoustic wave; The Brillouin spectroscopy system further comprises a spectrometer system; The spectrometer system comprises: a rubidium vapor absorbing filter that receives the first electromagnetic radiation and the second electromagnetic radiation and transmits the second electromagnetic radiation and attenuates the first electromagnetic radiation; a detector configured to receive a portion of the second electromagnetic radiation from the rubidium vapor absorbing filter without receiving the first electromagnetic radiation from the rubidium vapor absorbing filter, and to provide information related to a biomechanical property of the ocular tissue region; A Brillouin spectroscopic analysis system comprising:

14. The target electromagnetic spectrum is the wavelength of rubidium absorption lines.

14. The Brillouin spectroscopy system of claim 13.

15. The target electromagnetic spectrum is at a wavelength of 780 nm; 14. The Brillouin spectroscopy system of claim 13.

16. the spectral clean-up filter is configured to be locked to a wavelength of 780 nm; 14. The Brillouin spectroscopy system of claim 13.

17. The human interface includes: A fiber optic probe; A catheter; A pen-shaped endoscope At least one of the following is provided:

14. The Brillouin spectroscopy system of claim 13.

18. the laser light source is configured to be coupled to a computer system in communication with the human interface and the spectrometer system and configured to obtain a first biomechanical value of a first ocular tissue region of a patient, obtain a second biomechanical value of a second ocular tissue region of the patient, and identify a medical condition of the patient's ocular tissue by comparing the first biomechanical value to the second biomechanical value.

14. The Brillouin spectroscopy system of claim 13.

19. The computer system is further configured to assess axial asymmetry between the first ocular tissue region and the second ocular tissue region to identify the medical condition.

20. The Brillouin spectroscopy system of claim 18.

20. The computer system is further programmed to determine a Brillouin frequency f measured in dependence on an angle θ relative to each normal at the first eye tissue region and the second eye tissue region.

20. The Brillouin spectroscopy system of claim 19.

21. 1. A method of operating an apparatus for evaluating ocular tissue of a patient, comprising: obtaining a first biomechanical value of a first ocular tissue region of the patient using Brillouin spectroscopy; obtaining a second biomechanical value of a second ocular tissue region of the patient; and comparing the first biomechanical value to the second biomechanical value to identify a medical condition of the tissue of the eye of the patient; causing the device to execute the second biomechanical value being a biomechanical value obtained by an air puff optical imaging technique; comparing the first biomechanical value to the second biomechanical value includes assessing axial asymmetry between the first ocular tissue region and the second ocular tissue region to identify the medical condition; obtaining the first biomechanical value and obtaining the second biomechanical value includes determining a Brillouin frequency f measured in dependence on an angle θ relative to a normal at the first ocular tissue region and the second ocular tissue region; The Brillouin frequency f is f≒f ０ +Δfsinθ where f ０ is a value measured perpendicular to the first eye tissue region or the second eye tissue region, and Δf is the axial asymmetry between the first eye tissue region and the second eye tissue region. A method comprising:

22. said comparing includes determining a difference in elasticity in the patient's cornea; 22. The method of claim 21.

23. The medical condition includes keratoconus.

22. The method of claim 21.

24. the first ocular tissue region and the second ocular tissue region are located on the eye of the patient.

22. The method of claim 21.

25. The first biomechanical value is an elasticity value of the first ocular tissue region of the eye and the second biomechanical value is an elasticity value of the first ocular tissue region of the eye. the biomechanical value being an elasticity value of the second ocular tissue region of the eye.

22. The method of claim 21.

26. the first ocular tissue region is located in the eye of the patient and the second ocular tissue region is located in the other eye of the patient.

22. The method of claim 21.

27. the first biomechanical value being an elasticity value of the first ocular tissue region of the eye and the second biomechanical value being an elasticity value of a corresponding tissue region of the other eye; 22. The method of claim 21.

28. the first biomechanical value of the tissue of the patient's eye is a biomechanical value obtained at a central inferior region of the cornea of ​​the eye, and the second biomechanical value of the corresponding tissue of the other eye of the patient is a biomechanical value obtained at a central inferior region of the other eye.

22. The method of claim 21.

29. the first biomechanical value of the ocular tissue of the patient is a biomechanical value obtained at a corneal thinnest region of the eye; the second biomechanical value of the corresponding tissue of the other eye of the patient is a biomechanical value obtained at the thinnest region of the cornea of ​​the other eye; 22. The method of claim 21.

30. the first ocular tissue region comprises a site selected from the group consisting of corneal tissue, scleral tissue, and lens tissue; 22. The method of claim 21.

31. the second ocular tissue region comprises a site selected from the group consisting of corneal tissue, scleral tissue, and lens tissue; 22. The method of claim 21.

32. The second biomechanical value is a biomechanical value obtained using Brillouin spectroscopy.

22. The method of claim 21.

33. The air puff optical imaging technique is selected from the group consisting of Scheimpflug topography and optical coherence tomography.

22. The method of claim 21.

34. 1. A Brillouin spectroscopy system for evaluating tissue in an ocular tissue region of a patient, comprising: a tunable laser source configured to generate a first electromagnetic radiation having an electromagnetic spectrum; a vapor cell-based reference portion configured to capture a portion of the first electromagnetic radiation and generate a first error signal that includes information regarding a deviation of the electromagnetic spectrum from a target electromagnetic spectrum, the vapor cell-based reference portion generating the first error signal by monitoring absorption of the first electromagnetic radiation by atoms in the vapor cell-based reference portion; A laser light source system comprising: a human interface configured to deliver the first electromagnetic radiation to the ocular tissue region of the patient, the first electromagnetic radiation interacting with at least one acoustic wave endogenous to the ocular tissue region to generate at least one second acoustic wave based on the at least one acoustic wave; a human interface in which electromagnetic radiation is generated; a spectrometer system configured to receive a portion of the second electromagnetic radiation to provide information related to a biomechanical property of the ocular tissue region; a spectral clean-up filter configured to generate a second error signal that contains information about spectral noise in the first electromagnetic radiation; a controller configured to control a laser temperature of the laser light source based on the second error signal; A Brillouin spectroscopic analysis system comprising:

35. The target electromagnetic spectrum is the wavelength of the absorption line of an atomic species; 35. The Brillouin spectroscopy system of claim 34.

36. The target electromagnetic spectrum is the wavelength of rubidium absorption lines.

36. The Brillouin spectroscopy system of claim 35.

37. The target electromagnetic spectrum is at a wavelength of 780 nm; 37. The Brillouin spectroscopy system of claim 36.

38. the human interface comprises a fiber optic probe; 35. The Brillouin spectroscopy system of claim 34.

39. the fiber optic probe is a catheter; 39. The Brillouin spectroscopy system of claim 38.

40. The fiber optic probe is a pen-type endoscope.

40. The Brillouin spectroscopy system of claim 39.

41. the Brillouin spectroscopy system further comprises a computer system in communication with the human interface and the spectrometer system; The computer system is configured to obtain a first biomechanical value of a first ocular tissue region of a patient, obtain a second biomechanical value of a second ocular tissue region of the patient, and identify a medical condition of the tissue of the patient's eye by comparing the first biomechanical value to the second biomechanical value.

35. The Brillouin spectroscopy system of claim 34.

42. The computer system is further configured to assess axial asymmetry between the first ocular tissue region and the second ocular tissue region to identify the medical condition.

42. The Brillouin spectroscopy system of claim 41.

43. The computer system is further programmed to determine a Brillouin frequency f measured in dependence on an angle θ relative to each normal at the first eye tissue region and the second eye tissue region.

43. The Brillouin spectroscopy system of claim 42.

44. The Brillouin frequency f is f≒f ０ +Δfsinθ where f ０ is a value measured perpendicular to the first eye tissue region or the second eye tissue region, and Δf is the axial asymmetry between the first eye tissue region and the second eye tissue region.

44. The Brillouin spectroscopy system of claim 43.

45. the first error signal is generated by modulating the first electromagnetic radiation; 35. The Brillouin spectroscopy system of claim 34.

46. The Brillouin spectroscopy system further comprises at least one polarizer and at least one source of a magnetic field; the first error signal is generated by the polarization dependence of atomic absorption in the magnetic field; 35. The Brillouin spectroscopy system of claim 34.

Images